Abstract: Graft-versus-host disease (GvHD) remains the predominant factor limiting the widespread utilization of allogeneic stem cell transplantation (allo-SCT) for the treatment of patients with high-risk or recurrent hematological malignancies. The primary approach to the prevention of acute GvHD is the use of calcineurin inhibitors (CNI) with methotrexate. With this approach, approximately, 30-80% of patients undergoing matched related or unrelated stem cell transplantation will develop acute GvHD. For patients that develop acute GvHD, therapy has not changed in over 30 years and consists of systemic corticosteroids. This approach has substantial side-effects leading to severe long-term complications. Treatment for patients with steroid-refractory acute GvHD is suboptimal with fewer than 15% of patients treated living more than a year after diagnosis. Thus, new forms of therapy are badly needed to improve the outcome of patients undergoing allo-SCT. Aggressive therapy targeting donor T cells in patients with steroid refractory acute GvHD of the lower GI tract, has not improved the long term outcome of patients refractory to corticosteroid therapy. This has led to increased interest in understanding how conditioning therapy and GvHD alter the homeostatic environment of the lower GI tract. Over the past 36 months, my group has evaluated the function of a relatively new population of innate lymphoid cells, termed type 2 innate lymphoid cells (ILC2). These cells are found in the GI tract and generate IL-4, IL-5 and IL-13. The cytokines IL-25 and IL-33 are critical to the generation of ILC2 cells. In the current proposal, we demonstrate that ILC2 cells are radiation and chemotherapy sensitive, and that they poorly reconstitute over a three month period from donor bone marrow cells. We demonstrate that infusion of donor ILC2 cells can prevent and more importantly TREAT ongoing acute GvHD of the lower GI tract. This was associated with significant decreases in donor Th1/Th17 and Tc1 cells in the colon and small bowel and improvement in colonic epithelial cell integrity. The activity of ILC2 cells required the generation of IL-13 and amphiregulin (AREG) by the ILC2 cells. Administration of ILC2 cells had no effect on the GvL response. The goals of the current proposal are to assess the use of ILC2 cells as a novel approach to the treatment of acute GvHD. We will investigate the mechanism by which ILC2 cells treat lower tract GvHD, the function of myeloid derived suppressor cells (MDSCs) and Tregs in this activity, and the roles that IL-13, AREG, dendritic cells and intestinal subepithelial myofibroblasts (ISEMFs) have in the activity of ILC2 cells. We will demonstrate that ILC2 cells function in mice receiving CNI and/or steroids, which will allow us to rapidly translate these findings to patients. Finally, we will evaluate the mechanism for activity of ILC2 cells focusing on signaling downstream of IL-13/IL-13 receptor and NOTCH. Understanding how ILC2 cells function is critical to future clinical trials that will use human ILC2 cells to treat patients with steroid-refractory GvHD of the lower GI tract.